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Aim: The aim of this study was to determine reference values for oxygen saturation (SpO2 ) in neonates at mild and moderate altitudes.Methods: Our study included 41 097 consecutively born, asymptomatic neonates from 35 hospitals, located in Yunnan, China, with altitudes ranging from 267 to 2202 m. Pre-and post-ductal SpO2 of each neonate was measured at 24 hours of age and before hospital discharge. All study participants, according to the altitude of birth, were categorised into three groups: low (0-500 m), mild (500-1500 m) and moderate altitude (1500-2500 m).Results: Every 1000-m increase in altitude was associated with a 1.54 per cent decrease in mean SpO2 . The means of pre-ductal SpO2 at low, mild and moderate groups were 97.9%, 96.4% and 95.5%, respectively. We used the 2.5th percentile of SpO2 distribution as the cut-off for neonatal SpO2 screening and defined new cut-off values of ≤93% for mild altitudes, ≤92% for moderate altitudes and no adjustment for low altitudes.Conclusion: We recommend revised cut-off values for neonatal SpO2 at mild and moderate altitudes and provide new values for paediatricians to refer to when screening neonates for severe congenital heart or lung diseases. [ABSTRACT FROM AUTHOR]